Locomotion is an essential and evolutionarily-conserved behavior required for animals to navigate their environment. However, the precise neuronal networks underlying the genesis of locomotor activity remain unknown; elucidating these networks will help aid in the development of new therapies for spinal cord injury. Locomotion involves coordinated and alternating rhythmic activity between opposing limbs, as well as between antagonistic muscles of the same limb. The locomotor central pattern generator (CPG), a network of spinal interneurons, it thought to produce locomotion without supraspinal or sensory commands. Despite recent advances in the genetic identification of spinal interneurons, the specific neurons comprising the CPG remains unknown. Discovery of these neurons would represent a fundamental advance in the field. Intriguingly, although motor neurons serve as CNS output, direct stimulation of motor neuron axons, in the intact ex vivo neonate mouse spinal cord preparation, can trigger locomotor activity. This finding suggests that motor neurons may have access to the CPG through their centrally-projecting axon collaterals. Furthermore, optogenetic activation of excitatory glutamate-releasing spinal interneurons can evoke locomotor behavior during development. Collectively, these observations suggest that spinal axon collaterals of motor neurons may synapse with a set of yet unknown excitatory interneurons, which play a role in mediating locomotor activity. As motor neuron axon collaterals have a short span, tracking motor neuron axon collaterals provides an attractive tool to study locomotor circuitry. Preliminary evidence I have gathered has established a putative excitatory spinal interneuron class which may play this role. Through its own axon collaterals, I hypothesize that this novel interneuron class is a key mediator of locomotor activity. In Aim 1, I will study the spinal connectivity of this class of spinal interneurons through rabies viral tracing techniques combined with immunohistochemistry. In Aim 2, I will employ physiological and optogenetic assays to study its function in locomotor activity using a novel ex vivo preparation which I have developed. In summary, this comprehensive set of experiments will provide a solid foundation to further our understanding of spinal locomotor networks.